The genus Xanthomonas Dowson 1939 compromises a diverse group of gram-negative, obligate aerobic, non-fermentative, rods which are motile by a single polar flagellum, and can produce a brominated yellow pigment identified as xanthomonadin. All reported strains of this genus have been described as plant associated, and most are reported as being pathogenic to a particular plant host. Based on microbiological classification, this genus can be separated into at least five separate species: X. campestris, X. fragariae, X. ampelina, X. albilineans, and X. axonopodis. [Young, K. M., Due D. W., Bradbury J. F et al. 1978. A proposed nomenclature and classification for plant pathogenic bacteria. N. Z. J Agric Res. 21: 153-177; Vauterin, L., Yang, P., Hoste, B. et al. 1992. Taxonomy of xanthomonads from cereals and grasses, based on SDS-PAGE of proteins, fatty acid analysis and DNA hybridization. J. Gen. Microbiol. 138: 1467-1477].
In nature, these bacteria are—in principle—free-living—constituting a broad membership of both rhizospheric and phyllospheric microflora [Vauterin, L., Yang, P., Alverez A. et al. 1996. Identification of non-pathogenic Xanthomonas strains associated with plants. Syst. Appl. Microbiol. 19:96-105]. Many Xanthomonas spp. are known to be phytopathogenic, and can cause devastating agriculture diseases such as citrus canker in a variety of Citrus spp.; bacteria leaf spot of citrus; and leaf scaled disease in sugarcane [Bradbury, J. F. 1986. Xanthomonas Dowson 1939, 187. Pages 198-260. Bacteria. CAB International Mycological Institute, Slough, England].
Whereas a considerable number of publications exist regarding the genetic virulence factors associated with pathogenic species of Xanthomonas, a paucity exists within the literature regarding the principle mode of invasion and biofilm interactions for colonization. The most likely points of invasion of pathogenic strains of Xanthomonas spp. involve access via the stomata or through portals of injury to either the plant leaves or fruit [Zubrzycki, H. M. and Diamante, D. Z. A. 1987. Relationship between the amount of the inoculum and the infection caused by Xanthomonas campestris pv. citri on citrus seedlings through natural infections in the field. Pages 379-382 in: Proc. Int. Soc. Citriculture, Sao Paulo, Brazil; and Schubert T. S., Rizvl, S. A. Sun, X et al. 2001. Meeting the challenge of eradicating citrus canker in Florida—Again. Plant Disease 85: 340-356]. However, natural invasion through stomata may require a higher inoculum source. Such concentrations are higher than what normally could be achieved under a healthy phyllosphere condition [Pruvost, O., Boher, B., Brocherieux, M., et al. 2001. Survival of Xanthomonas axonopodis pv. citri in leaf lesions under tropical environmental conditions and simulated splash dispersal of inoculum. Phytopathology 92: 336-346]. Thus—not being delimited by theory—the principle mode of invasion of such organisms may be primarily due through injured areas of the plants. Such injuries could occur via mechanical means or through insect engendered injury.
Current methods of controlling plant infections by Xanthomonas spp. include the use of copper containing sprays. Likewise such sprays have been utilized to cosmetically change the appearance of canker lesions on certain citrus fruits. However, the use of such sprays is delimiting due to their toxicity. Furthermore, some Xanthomonas spp. have become resistant to such copper controlling agents [Gardan, L., Brault, T., and Germain E. 1993. Copper resistance of Xanthomonas campestris pv. juglands in French walnut orchards and it association with conjugative plasmids. Acta Hort. (ISHS) 311:239-265]. Thus, a priori conclusions suggests that many Xanthomonas spp. may express resistance to copper sprays. This may not be exclusive of X. axonopodis [pv.] citri—the etiological agent of citrus canker. Furthermore, it has been suggested that utilization of such spraying methods may exacerbate the spread of citrus bacterial spot [Gottwald, T. R., Graham, J. H., and Riley T. D. 1997. The influence of spray adjuvants on exacerbation of citrus bacterial spot. Plant Disease. 81:1305-1210].
Most states have adopted considerable eradication programs regarding the control of xanthomonad-related diseases. None is more prevalent than Florida's eradication program targeting citrus canker. Regulated decontamination using a variety of agents is strictly enforced, and Florida incorporates the removal and destruction of infected trees along a specific radius of the infected areas. One concern regarding such methods is that the method of removing such infected trees may aid in the dispersal of the bacterium. Nonetheless, control measures have had limited success and are very expensive to both regulatory bodies and citrus grove owners. Thus alternative measures for expedient and economical control of such diseases are justified.
In nature, many rhizosphere and phyllosphere microflora express characteristics which keep some pathogenic bacteria species in a balance via competition for nutrients or the release of factors which injure or kill possible pathogens. Such characteristics are focused under the concept of biological control. Since the early 1990's, considerable efforts have focused on the control of fungal and bacterial disease using organisms which excrete killing agents directed at such pathogens, or organisms which more fastidiously compete for nutrients against the proposed pathogen—thereby competitively retarding the growth of the more pathogenic species.
Whereas the concept of biological control has been effective in controlling many fungal diseases, biological control has just recently began to focus on xanthomonad-related infections. For example, certain bacterial eating protozoa have been identified within the rhizosphere which control certain pathogenic Xanthomonas spp. within the soil in proximity to the host plant [Habte, M. and Alexander, M. 1975. Protozoa as agents responsible for the decline of Xanthomonas campestris in soil. Appl. Microbiol. 29(2): 159-164]. To date, such organisms have not been employed in fighting xanthomonad-related diseases. In contrast, xanthomonads have been employed as biological control agents to control noxious weeds. Xanthomonas campestris pv. poae is utilized as a biological control agent of annual bluegrass [Imaizumi, S., Tateno, A., Morita, K., Fujimori, T. 1999. Seasonal factors affecting the control of annual bluegrass (Poa annua) with Xanthomonas campestris pv. poae. Biol. Control 16:18-26].
In some cases, invasion of phytopathogenic microbes themselves induce defense mechanisms within plants to prevent further injury. The vast majority of injuries in some agriculture crops do not become infected because of the plant's own defense system. A considerable number of publications present convincing evidence that some rhizosphere microorganisms can induce systemic resistance in plants against root and foliar diseases. For example, the plant growth-promoting rhizobacteria Pseudomonas fluorescens and Serratia marcescens can induce systemic resistance in cucumber to anthacnose [Wei, G., Kloepper, J. W., and Tuzun, S. 1991. Induction of systemic resistance of cucumber to Colletotrichum orbiculare by select strains of plant growth promoting rhizobacteria. Phytopathology 81:1508-1512], and the cucumber mosaic virus [Raupach, G. S., Liu, L. Murphy K. F, et al. 1996. Induced systemic resistance in cucumber and tomato against cucumber mosaic cucumovirus using plant growth-promoting rhizobacteria. Plant Disease. 80:891-894].
Capitalizing on the induction of plant defense mechanisms, extractions from a variety of non-plant-pathogenic microorganisms—fungi and bacteria primarily involved with composting—have been employed to fight a variety phytopathogenic microbes under the premise of inducing resistance following exposure to extracts of non-phytopathogenic microbes. Such formulations have been described in U.S. Pat. No. 6,326,016.
In fact, metabolites from microorganisms associated in the production of compost tea have been readily effective in controlling some of the most devastating Xanthomonas spp. related diseases such as citrus canker. This has been more successful as a preventative measure in healthy citrus trees [Ozores-Hampton, M. O. and Obreza, T. A. 2000. A composted waste use on Florida crops: A Review. International Composting Symposium, Nova Scotia]. Furthermore, evidence exists which suggests that Pantoea agglomerans strain E278Ar can induce systemic resistance against X. campestris pv. armoraciae induced leaf spot disease in radish [Han, D., Y., Colin, D. L., Bauer, W. D., and Hoitink, H. A. J. 2000. A rapid bioassay for screening rhizosphere microorganisms for their ability to induce systemic resistance. Phytopathology 90:327-332].
As early as 1991, a considerable number of studies have focused on the actions of such microorganisms to produce resistance of plants toward specific phytopathogens. The type of systemic resistance induced by nonpathogenic microorganism exposure to plants has been termed induced systemic resistance (ISR) [Kloepper, J. W., Tuzun, S., and Kuć, J. A. 1992. Proposed definitions related to induced disease resistance. Biocontrol Sci. Technol. 2:349-351]. This is different from systemic acquired resistance (SAR) which is induced by necrotizing plant pathogens [Delaney, T. P. 1997. Genetic dissection of acquired resistance to disease. Plant Physiol. 113: 5-12; and Ryals, J., Neuenschwander, U. H., Willits, M. G. et. al. 1994. Systemic acquired resistance. Plant Cell 8:1809-1819].
Based on this rapidly growing field of understanding both ISR and SAR, several synthetic compounds have been developed which elicit plant defense reaction. One such compound, acibenzolar-S-methyl (Actigard 50W, Bion 50WG, Syngenta, Basel Switzerland) has been developed. Such compounds have demonstrated limited success in controlling X. axonopodis pv. vesicatoria induced bacterial spot disease in tomatoes [Louws, F. J., Wilson, M., Campbell, H. L., et. al. 2001. Field control of bacterial spot and bacterial speck of tomato using a plant activator. Plant Disease. 85: 481-488]. Other activators do exist. However, a major concern regarding the use of synthetic activators is the long-term phytotoxicity and bioactivity in controlling the induction of disease resistance over time in field application.
Natural activators of plant resistance exist. Once such factor is riboflavin. Riboflavin has been demonstrated to act as an effective elicitor for systemic resistance, and as an activator in plants by inducing the a signal transduction pathway for the expression of pathogenesis-related genes [Dong, H. and Beer, S. V. 2000. Riboflavin induces disease resistance in plants by activating a novel signal transduction pathway. Phytopathology 90: 801-811]. Direct application of riboflavin as an elicitor for systemic resistance has been limited to laboratory experiments. Its exogenous use in the field on a variety of plants has not been set into formal practice at this time.
Likewise, silicon (Si2) has been investigated regarding its role in initiating plant resistance to disease. Such actions are controversial [Werner D., and Roth R. 1983. Silica metabolism. Pages 683-694 in: Inorganic Plant Nutrition. A. Läuchi and R. L. Bieleski, eds. Sringer-Verlag, Berlin; and Epstein, E. 1994. The anomaly of silicon in plant biology. Proc. Natl. Acad. Sci. U.S.A. 91: 11-17]. However, such controversy revolves around passive mechanical protection of plants. Nonetheless, Si2 has been shown to be effective in protecting certain dicots against fungal disease and demonstrate some aspect of SAR toward such invading pathogens [Bélanger, R. R., Bowen, P. A., Ehret, D. L. et al. 1995. Soluble silicon: Its role in crop and disease management of green house crops. Plant Disease. 79:329-336].
Moreover, Fawe et al. have provided evidence which suggest that Si2 may offer protection of cucumber from Pythium ultimum by inducing cucumber production of the phytoalexins such as flavonol aglycone rhamnetin [Fawe, A., Abouu-Zaid, M., Menzies, J. G. et al. 1998. Silicon-mediated accumulation of flavonoid phytoalexins in cucumber. Phytopathology 88:396-401].
Despite the controversy surrounding the use of such agents for adding protection of plants from disease, the skilled practitioner can identify that such agents can be added to this invention. However, extracts used in our product from Y. schidigera are known to posses many plant growth promoting and plant protection stimulants [Zielgler, D. M. 1990. Flavin-containing monooxygenases: enzymes adapted for multisubstrate specificity. Trends in Pharmacol. Sci. 11: 321-324; Ziegler, D. M. 1993. Recent studies on the structure and function of multisubstrate flavin-containing monooxygenases. Ann. Rev. Pharmacol. Toxicology 33: 179-199; and Zhoa, Y., Christensen, S. K., Frankhauser, C., et al. 2001. A role for flavin monoxygenase-like enzymes in auxin biosynthesis. Science 291:306-309].
In relation to biological control, many Trichoderma spp. have been employed as biological control agents toward plant fungal diseases and have demonstrated the ability to elicit SAR to such pathogens. One such elicitor factor from the fungus Trichoderma virens has been identified and described in U.S. Pat. No. 6,242,420. However its field application is currently unresolved, and in principle it is directed toward fungal plant diseases associated with the rhizosphere.
Many fungi of the genus Trichoderma produce substances which kill or inhibit the growth of a variety of aerobic and anaerobic bacteria. Based on this principle, T. virens has been used as a mycoparasite and antibiotic-producing antagonist of plant pathogens, and has demonstrated to be an effective biological control agent against a variety of soil-borne root diseases [Reyes A. A. 1985. Suppression of Fusarium and Pythium pea root rot by antagonistic microorganisms, Phytoprotect. 66:23-29]. Furthermore, intact cultures and spent culture medium from T. virens have been used as biological control agent against many phytopathogenic fungi as described by Tahvonen et al. in U.S. Pat. No. 5,968,504. A major problem in using this organism is based on the stability of its formulation; whereby, the viability of this organism is affected. Such formulations have been described in U.S. Pat. No. 4,724,147. Likewise, extracts from Trichoderma spp. have been employed with other added microbial extracts to induce resistance in plants against phytopathogenic microbes as described in U.S. Pat. No. 6,326,016.
Trichoderma spp. produce a considerable number of agents regarded as antibiotics which include gliotoxin, viridin, gliviron, and heptelidic acid. Heptelidic acid has been shown to be an effective antibiotic and antifungal agent. [Ghisalberti, E. L. and Sivasithamparan, K. 1991. Antifungal antibiotics produced by Trichoderma spp. Soil Biol. Biochem. 23:1011-1021].
Many Trichoderma spp. produce a variety of products which have profound antibiotic effects. One such class of metabolites are designated as peptaibols. These are short chain polypeptides ranging from 15 to 20 amino acids. Most of their amino acids are atypical such as hydroxyproline, isovaline, ethylnorvaline, and aminioisobutyric acid. As the name implies, peptaibols are generally regarded as protein antibiotics. The antimicrobial activity of peptaibols is thought to result from the ability to disrupt the integrity of lipid membranes—thus being more effective against gram-negative bacteria and fungi. To date, the most characterized peptaibols are disclosed from the fungi genera of Trichoderma and Emericellopsis. 
In concern of this invention, peptaibols from T. harzianum include trichorzianins, trichokindins, trichorzines, and harzianins [El Hajji, M., Rebuffat, S., Lecommandeur, D., and Dodo, B. 1987. Isolation and sequence determination of trichorzianines A antifungal peptides from Trichoderma harzianum. Int J Peptide Protein Res. 29:207-215; Iida A, Sanekata, M., Fugita, T. et al. 1987. Fungal metabolites XVI. Structure of new peptaibols, trichorzins I-VI from the fungus Trichoderma harzianum. Int J Peptide Protein Res. 29:207-215; Rebuffat, S., El Hajji, M., Henning, P. et al. 1989. Isolation sequence, and conformation of seven trichorzianines B from Trichoderma harzianum. Int J Peptide Protein Res. 34:200-210; and Sawa, R., Mori, Y., Inuma, H. 1994. Harzianic acid, a new microbial antibiotic from a fungus. J. Antibiotics (Tokyo) 47: 731-732]. This invention capitalizes on the production of such metabolites from T. harzianum. 
Trichoderma harzianum strains have been used as biological control agents against nematodes and fungal diseases in certain plants, and have been indicated as a plant promoting agent as cited in U.S. Pat. No. 6,475,772. Likewise, certain strains of T. harzianum have been exempted from requirements of a temporary tolerance and are employed as biological control agents for rhizosphere related fungal diseases in agricultural crops [Hasan, S. B. 2000. Trichoderma harzianum Rifai Strain T-39; Exemption from the Requirements of a Tolerance, Federal Register 65:121, pp. 38753-38757]. Nevertheless, a paucity exists within the literature regarding its applications to phyllosphere pathogens—in particular toward pathogens belonging to the genus Xanthomonas. 
Employment of Trichoderma spp. as a biological control agent has been more successful against fungal phytopathogens. The major mechanism accounting for such actions include their microparasitism of both the invading pathogen and plant host evoking a cascade of events including the plant host cell protective interactions and direct degradation of the targeted fungal pathogen by the Trichoderma spp. employed.
Secreted chitanases and glucanases (β-1,3-glucanase and β-1,6-glucanase), secreted by Trichoderma spp. have been attributed to be involved in not only degrading other mycoparasites, but may play a role in eliciting plant cell defense mechanisms. Whereas such glucanases have been reported to lyse fungal and yeast walls, such enzymes have also been reported to lyse bacteria. [De La Cruz, J, Pintor-Toro, J. A., Benitez, T., and Llobell, A. 1995. Purification and characterization of an endo-β-1,6-glucanase from Trichoderma harzianum that is related to its mycoparasitism. J. Baceriol. 177: 1864-1871]. Nevertheless, the role of chitanases, β-1,3-glucanase and β-1,6-glucanase in inducing host cell defense responses remain obscure.
A major problem with the use of many Trichoderma spp. is that they produce phytotoxins which can injure targeted host plants. One such toxin is viridin [Jones, R. W., W. T. Lanini, and J. G. Hancock. 1988. Plant growth response to the phytotoxin viridiol produced by the fungus Gliocladium virens. Weed Sci. 36:683-687; Weindling, R., and O. H. Emerson. 1936. The isolation of a toxic substance from the culture filtrate of Trichoderma. Phytopathology 26:1068; and Howell, C. R., and R. D. Stipanovic. 1984. Phytotoxicity to crop plants and herbicidal effects on weeds of viridiol produced by Gliocladium virens. Phytopathology 74:1346-1349]. In this respect, T. harzianum, also produces viridin.
Viridin is associated with sterol production in such fungi, and in 1994, Howell and Stipanovic demonstrated that viridin production in Gliocladium virens was significantly reduced when the fungus was grown in the presence of added steroid inhibitors. Likewise, such fungi not only demonstrated a reduction in viridin (as viridiol) production, but had decreased the phytotoxicity of this species [Howell, C. R., and Stipanovic R. D. 1994. Effect of sterol biosynthesis inhibitors on phytotoxin (viridin) production by Gliocladium virens in culture. Phytopathology 84:969-972]. This process of inducing the inhibition of viridiol is described in more detail in U.S. Pat. No. 5,882,915. In reference to this invention, the skilled microbiologist can quickly identify that such methods could be employed in this present invention. However, due to the extraction process employed in the present invention, recovered products from T. harzianum contain very little—if any—viridin and viridiol.
In reference to this invention, we originally used extracts from a strain of T. harzianum obtained from Venture Biodiscovery, Largo, Fla. This strain, MBMH-21 was first isolated from soil samples in Mission, Tex. However, with regards to this invention, we have found that a variety of T. harzianum strains work well. Such strains employed have included T. harzianum (American Type Culture Collection (ATCC) No. 20873). Other strains readily available from many distributers and fresh soil isolates of T. harzianum have also been employed in this invention.
The use of bioextracts and/or intact viable T. harzianum (or other Trichoderma spp.) are based on the phenomenon of induced systemic resistance as described above. As described above, such occurrence has been observed when nonpathogenic organisms are introduced into a host which triggers a cascade of defenses to fight off the invasions of pathogenic organisms. This stands to reason that exposure of a plant to a related non-phytopathogenic species may develop ISR to the related phytopathogen. In relation to this invention, we apply extracts from nonpathogenic Xanthomonas spp. Such employment is provided below in the detailed description of the invention. In relation to this invention, we employ extracts from X. theicola and X. codiaei. These organisms can be isolated from soil and certain plants. Furthermore, they can be purchased from such depository agencies as the ATCC.
Another component of our invention are extracts from the plant Yucca schidigera. Included in these extracts are saponins. Saponins are natural detergents found in many plants. Saponins have detergent or surfactant properties because they contain both water-soluble and fat soluble components. They consist of a fat-soluble nucleus, having either a steroid or triterpenoid structure, with one or more side chains of water-soluble carbohydrates. Saponins have been employed in a variety of food products, livestock feed, cosmetics, and beverages. Likewise, they have been indicated in therapeutic value in treating a variety of diseases [Bingham, R., Bellew, B. A., Bellew J. G. 1975. Yucca plant saponin in the management of arthritis. J Appl Nutr. 27:45-50; Hartwell, J. L. 1976. Types of anticancer agents isolated from plants. Cancer Treat Rep. 60:1031-1067; Scarpato, R., Bertoli, A., Baccarati A., et al. 1998. Different effects of newly isolated saponins on the mutagenicity and cytotoxicity of the anticancer drugs mitomycin C and bleomycin in human lymphocytes. Mutat. Res. 420: 49-54].
Saponins have been employed as synergist in combination with aldehydes to control plant and animal pathogens as described in U.S. Pat. No. 5,639,794. Extracts from Y. schidigera and Y. hedera have been effective in controlling non-aquatic snails and slugs as identified in U.S. Pat. No. 5,290,557. It is evident that addition of saponins aid admixtures directed in controlling fungal and pest diseases among a variety of plants as cited within U.S. Pat. Nos. 5,639,794 and 6,482,770. However, to our knowledge, this invention is the first to apply Y. schidigera extracts (containing saponins and other against) against xanthomonad-related plant/crop diseases.
A major component to this invention involves principle extracts from certain Xanthomonas spp. Specific species employed in our invention include X. theicola and X. codiaei. Employment of these organisms is based on principles observed with the use of the outer membrane fractions of P. fluorescens to induce ISR in agriculture crops as described above. Both animal and plant host interactions with gram-negative bacteria pathogens are dependent on a cascade of events. Lipopolysaccharides are extremely effective in inducing both hypersensitivity reactions as well as ISR. The skilled practitioner will recognize that such extracts from more closely related targeted host specific diseases may be employed. However, our studies have provided evidence which suggest that the extracts from X. theicola and X. codiaei evoke hypersentitivity reactions (HR) in a variety of plants [i.e. citrus, tomatoes, and sugarcane]. Furthermore, the use of these relatively safe nonpathogenic Xanthomonas spp. are more environmentally friendly regarding possible transformation into an infectious phytopathogen.